Mutant phosphoribosylpyrophosphate synthetase and method for producing L-histidine

ABSTRACT

The present invention relates to a mutant bacterial PRPP synthetase which is resistant to feedback by purine nucleotides, and a method for producing L-histidine using the bacterium of the Enterobacteriaceae family wherein the L-amino acid productivity of said bacterium is enhanced by use of the PRPP synthetase which is resistant to feedback by purine nucleotides, coded by the mutant prsA gene.

This application claims the benefit of provisional application60/587,492, filed Jul. 14, 2004.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a method for producing L-amino acid,such as L-histidine. More specifically, the present invention relates toa novel feedback-resistant enzyme involved in the biosynthesis ofpurines and L-histidine. More specifically, the present inventionconcerns a new feedback-resistant mutant phosphoribosylpyrophosphatesynthetase (PRPP synthetase) from E. coli. The invention also relates toa method for producing L-histidine by fermentation using bacterialstrains containing the novel feedback-resistant enzyme.

2. Background Art

Conventionally, L-amino acids are industrially produced by fermentationmethods utilizing strains of microorganisms obtained from naturalsources or mutants thereof, which are modified to enhance productionyields of L-amino acids.

Many techniques to enhance production yields of L-amino acids have beenreported, including transformation of microorganisms with recombinantDNA (see, for example, U.S. Pat. No. 4,278,765). Other techniquesinclude increasing the activities of enzymes involved in amino acidbiosynthesis and/or desensitizing the target enzymes of the feedbackinhibition by the resulting L-amino acid (see, for example, JapaneseLaid-open application No. 56-18596 (1981), WO 95/16042 or U.S. Pat. Nos.5,661,012 and 6,040,160).

5-Phosphoribosyl-α-1-pyrophosphate (hereinafter, “PRPP”) andadenosine-5′-triphosphate (hereinafter, “ATP”) are the initialsubstrates in histidine biosynthesis. PRPP can sometimes induce thehistidine biosynthesis to follow divergent pathways, resulting in thebiosynthesis of pyrimidine nucleotides, purine nucleotides, pyridinenucleotides, and tryptophan (Escherichia coli and Salmonella, SecondEdition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C.,1996).

Many nucleotides competitively inhibit the activity of PRPP synthetasewith ATP. However, the only potent nucleotide inhibitor isadenosine-5′-diphosphate (ADP); it competes with ATP and is anallosteric inhibitor that binds to a site other than the active site(Hove-Jensen, B. et al, J. Biol. Chem. 261:6765-6771 (1986)).

Mutants with altered PRPP synthetase have been obtained in both E. coliand S. typhimurium. One of the E. coli mutants produces a PRPPsynthetase with a 27-fold increase in the K_(m) value for ATP, and theenzyme is no longer inhibited by AMP. This mutation results fromsubstitution of aspartic acid 128 by alanine (prsDA mutation). One S.typhimurium prs mutant is temperature-sensitive and has only 20% of thewild-type PRPP synthetase activity. This mutant enzyme had elevatedK_(m) values for ATP and ribose 5-phosphate and reduced sensitivity toinhibition by ADP. The mutation is the result of the replacement ofarginine 78 by cysteine (Escherichia coli and Salmonella, SecondEdition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C.,1996).

It is well known that superactivity of human PRPP synthetase andresistance to purine nucleotide are associated with neurodevelopmentalabnormalities in addition to hyperuricemia and gout (Becker M. A. et al,Arthritis Rheum, 18:6 Suppl: 687-94 (1975); Zoref E. et al, J. Clin.Invest., 56(5): 1093-9 (1975)). Uric acid overproduction in individualswith superactivity of PRPP synthetase results from increased productionof PRPP and consequent acceleration of purine nucleotide synthesis denovo. It was shown that superactivity of PRPP synthetase is a result ofan A to G mutation at nucleotide 341, which results in an asparagine toserine substitution at amino acid residue 113 of the mature enzyme. Thismutant PRPP synthetase is resistant to purine nucleotides that inhibitthe normal enzyme by a mechanism that is noncompetitive with respect toATP (Roessler, B. J. et al. J. Biol. Chem., v. 268, No 35, 26476-26481(1993); Becker, M. A. et al, J. Clin. Invest., 96(5): 2133-41 (1995)).

A process for producing purine nucleosides via fermentation of amicroorganism belonging to the genus Escherichia and having purinenucleoside-producing ability, and containing a prsDA mutation isdisclosed (European patent application EP1004663A1). However, there areno reports describing mutant bacterial PRPP synthetase which isfeedback-resistant to purine nucleotides, or the use of such a mutantPRPP synthetase for improving L-histidine production usingL-histidine-producing strains.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a new mutant bacterialPRPP synthetase. Furthermore, it is an object of the present inventionto provide an L-histidine-producing strain containing the mutant PRPPsynthetase, which has enhanced production yields of L-histidine. Also,it is an object of the present invention to provide a method forproducing L-histidine using the above-described strain.

It is an object of the present invention to provide a mutant bacterialphosphoribosylpyrophosphate synthetase (PRPP synthetase), wherein theaspartic acid at position 115 in a wild-type phosphoribosylpyrophosphatesynthetase from Escherichia coli is substituted with another L-aminoacid residue, and feedback inhibition by purine nucleotides isdesensitized.

It is a further object of the present invention to provide the mutantPRPP synthetase described above, wherein the aspartic acid residue atposition 115 in a wild-type PRPP synthetase is substituted with anserine residue.

It is a further object of the present invention to provide the mutantPRPP synthetase as described above, wherein the wild-type PRPPsynthetase is derived from Escherichia coli.

It is a further object of the present invention to provide the mutantPRPP synthetase as described above, which includes deletion,substitution, insertion, or addition of one or several amino acids atone or a plurality of positions other than position 115, whereinfeedback inhibition by purine nucleotides is desensitized.

It is a further object of the present invention to provide a DNAencoding a mutant PRPP synthetase as described above.

It is a further object of the present invention to provide a bacteriumof the Enterobacteriaceae family, which contains the DNA describedabove, and has an ability to produce L-histidine.

It is a further object of the present invention to provide the bacteriumas described above, wherein the activity of the mutant PRPP synthetaseis enhanced.

It is a further object of the present invention to provide the bacteriumas described above, wherein the bacterium belongs to the genusEscherichia.

It is a further object of the present invention to provide the bacteriumas described above, wherein the activity of the mutant PRPP synthetaseis enhanced by increasing the expression of the mutant PRPP synthetasegene.

It is a further object of the present invention to provide the bacteriumas described above, wherein the activity of the mutant PRPP synthetaseis enhanced by increasing the copy number of the mutant PRPP synthetasegene, or modifying an expression control sequence of the gene so thatthe expression of the gene is enhanced.

It is a further object of the present invention to provide the bacteriumas described above, wherein the copy number is increased by integrationof additional copies of the mutant PRPP synthetase gene into thechromosome of the bacterium.

It is a further object of the present invention to provide a method forproducing L-histidine comprising cultivating the bacterium as describedabove in a culture medium, allowing the L-histidine to accumulate in theculture medium, and collecting the L-histidine from the culture medium.

It is a further object of the present invention to provide the method asdescribed above, wherein the bacterium has enhanced expression of thegenes for histidine biosynthesis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The above-described objects were achieved by constructing a new mutantPRPP synthetase from E. coli. Based on the high conservatism of the prsAgene (Taira M. et al., J. Biol. Chem., v. 262, No 31, pp.14867-14870(1987)), the mutant E. coli PRPP synthetase having a mutationcorresponding to the human mutation Asn-113 was constructed. It wasshown that the use of such a mutant PRPP synthetase enhances L-histidineproduction when additional copies of the gene encoding the mutant PRPPsynthetase are introduced into a L-histidine-producing strain. Thus, thepresent invention has been completed.

(1) Mutant PRPP Synthetase and Mutant prsA Gene.

The mutant PRPP synthetase of the present invention is referred to as“the mutant PRPP synthetase” hereinafter and is defined as having asubstitution at the aspartic acid residue at position 115 of wild-typePRPP synthetase. A DNA coding for the mutant PRPP synthetase is referredto as “the mutant prsA gene” or “mutant PRPP synthetase gene,” and aPRPP synthetase without the above position 115 substitution is referredto as “wild-type PRPP synthetase.”

It is known that the genetic and functional basis of superactivity ofhuman PRPP synthetase associated with resistance to purine nucleotide iscaused by single base substitution in prsA gene (Roessler, B. J. et al.J. Biol. Chem., v. 268, No 35, 26476-26481 (1993)). Based on the highconservatism of prsA gene (Taira M. et al., J. Biol. Chem., v. 262, No31, pp.14867-14870 (1987)), the mutant PRPP synthetase from E. colihaving a mutation corresponding to the human Asn-113 mutation wasconstructed. This mutation has never been described for all bacterialPRPP synthetases. The phrase “bacterial PRPP synthetase” means the PRPPsynthetase existing in the bacteria of Enterobacteriaceae family,corynebacteria, bacteria belonging to the genus Bacillus etc.Enterobacteriaceae family includes bacteria belonging to the generaEscherichia, Erwinia, Providencia and Serratia. The genus Escherichia ispreferred.

The substitution of the aspartic acid at position 115 of wild-type PRPPsynthetase [EC 2.7.6.1] from E. coli with any amino acid, preferablywith serine, leads to formation of a mutant protein which isfeedback-resistant to purine nucleotides, such as purine di- andmononucleotides, mainly guanosine-5′-diphosphate (GDP),adenosine-5′-diphosphate (ADP) and adenosine-5′-monophosphate (AMP).

The mutant PRPP synthetase can be obtained by introducing mutations intoa wild-type prsA gene using known methods. The prsA gene of E. coli(nucleotide numbers 1260151 to 1261098 in the sequence of GenBankAccession NC_(—)000913, gi: 16129170, SEQ ID NO: 1) is one example of awild-type prsA gene. The prsA gene is located between the ychM and ychBORFs on the chromosome of E. coli strain K-12. Therefore, the prsA genecan be obtained by PCR (polymerase chain reaction; see White, T. J. etal., Trends Genet., 5, 185 (1989)) utilizing primers prepared based onthe nucleotide sequence of the gene. Genes coding for PRPP synthetase ofother microorganisms can be obtained in a similar manner.

The mutant PRPP synthetase may include deletion, substitution,insertion, or addition of one or several amino acids at one or aplurality of positions other than 115, provided that the activity ofPRPP synthetase is not lost. The phrase “activity of PRPP synthetase”means an activity catalyzing the reaction of ribose-5-phosphate and ATPwith release of AMP to form 5-phosphoribosyl-α-1-pyrophosphate (PRPP).The PRPP synthetase activity of the extracts and degrees of inhibitionby ADP can be measured using the partially modified method of K. F.Jensen et al. (Analytical Biochemistry, 98, 254-263 (1979)).Specifically, [α-³²P]ATP can be used as the substrate and [³²P]AMPproduced by the reaction should be measured.

The number of “several” amino acids differs depending on the position ortype of amino acid in the three dimensional structure of the protein.This is because some amino acids have high homology to one another andtherefore do not greatly affect the three dimensional structure of theprotein. Therefore, the mutant PRPP synthetase of the present inventionmay be one which has homology of not less than 30 to 50%, preferably 50to 70%, more preferably 70% to 90%, and most preferably 95% or more,with respect to the entire PRPP synthetase amino acid sequence, andwhich retains the PRPP synthetase activity.

In the present invention, “position 115” means position 115 in the aminoacid sequence of SEQ ID NO: 2. In the PRPP synthetase from E. coli, theamino acid residue in position 115 is aspartic acid. A position of anamino acid residue may change, for example, if an amino acid residue isinserted at the N-terminus portion, the amino acid residue inherentlylocated at position 115 becomes position 116. In this situation, theamino acid residue corresponding to the original position 115 is to meanthe amino acid residue at position 115 in the present invention.

To determine the L-amino acid residue corresponding to position 115 ofPRPP synthetase from E. coli, it is necessary to align the amino acidsequence of PRPP synthetase from E. coli (SEQ ID NO: 2) and an aminoacid sequence of PRPP synthetase from the bacterium of interest.

The DNA of the present invention, which codes for the substantially thesame protein as the mutant PRPP synthetase described above, may beobtained, for example, by modifying the nucleotide sequence, forexample, by means of site-directed mutagenesis so that one or more aminoacid residues at a specified site are deleted, substituted, inserted, oradded. The DNA modified as described above may be obtained byconventionally known mutation treatments. Mutation treatments include amethod for treating in vitro a DNA containing the mutant prsA gene, forexample, with hydroxylamine, and a method for treating a microorganism,for example, a bacterium, belonging to the genus Escherichia containingthe mutant prsA gene with ultraviolet irradiation or a mutating agentusually used for the such treatment, such asN-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.

The substitution, deletion, insertion, or addition of a nucleotide asdescribed above also includes mutation, which naturally occurs (mutantor variant), for example, on the basis of the individual difference orthe difference in species or genus of bacterium, which contains PRPPsynthetase.

The DNA, which codes for substantially the same protein as the mutantPRPP synthetase, can be obtained by isolating a DNA which hybridizes asa probe to DNA having a known prsA gene sequence or part of it, understringent conditions, and which codes for a protein having the PRPPsynthetase activity. The DNA may be isolated from a cell containing themutant PRPP synthetase which is subjected to mutation treatment.

The phrase “stringent conditions” in the present invention meansconditions under which so-called specific hybrids are formed, andnon-specific hybrids are not formed. It is difficult to express thiscondition precisely by a numerical value. However, for example,stringent conditions include conditions under which DNAs having highhomology, for example, DNAs having homology of not less than 50% witheach other hybridize to each other, and DNAs having homology lower thanthe above will not hybridize with each other.

To evaluate the degree of protein or DNA homology, several calculationmethods, such as BLAST search, FASTA search and CrustalW, can be used.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastp, blastn, blastx, megablast,tblastn, and tblastx; these programs ascribe significance to theirfindings using the statistical methods of Karlin, Samuel and Stephen F.Altschul (“Methods for assessing the statistical significance ofmolecular sequence features by using general scoring schemes”. Proc.Natl. Acad. Sci. USA, 1990, 87:2264-68; “Applications and statistics formultiple high-scoring segments in molecular sequences”. Proc. Natl.Acad. Sci. USA, 1993, 90:5873-7). FASTA search method described by W. R.Pearson (“Rapid and Sensitive Sequence Comparison with FASTP and FASTA”,Methods in Enzymology, 1990 183:63-98). ClustalW method described byThompson J. D., Higgins D. G. and Gibson T. J. (“CLUSTAL W: improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrixchoice”, Nucleic Acids Res. 1994, 22:4673-4680).

Alternatively, stringent conditions are exemplified by conditions underwhich DNAs hybridize with each other at a salt concentrationcorresponding to ordinary conditions of washing in, Southernhybridization, i.e., 60° C., 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1%SDS. As a probe for the DNA that codes for variants and hybridizes withprsA gene, a partial sequence of the nucleotide sequence of SEQ ID NO: 1can also be used. Such a probe may be prepared by PCR usingoligonucleotides based on the nucleotide sequence of SEQ ID NO: 1 asprimers, and a DNA fragment containing the nucleotide sequence of SEQ IDNO: 1 as a template. When a DNA fragment of about 300 bp is used as theprobe, the washing conditions for the hybridization consist of, forexample, 50° C., 2×SSC, and 0.1% SDS. Duration of the washing proceduredepends on the type of membrane used for blotting and, as a rule, isrecommended by manufacturer. For example, recommended duration ofwashing the Hybond™ N+ nylon membrane (Amersham) under stringentconditions is 15 minutes.

The gene, which is hybridizable under conditions as described above,includes those having a stop codon generated within a coding region ofthe gene, and those having no activity due to mutation of the activecenter. However, such inconveniences can be easily removed by ligatingthe gene with a commercially available expression vector, andinvestigating the PRPP synthetase activity of the expressed protein.

(2) Bacterium of the Present Invention.

The bacterium of the present invention is an L-histidine-producingbacterium of the Enterobacteriaceae family containing DNA encoding themutant PRPP synthetase of the present invention. Furthermore, thebacterium of the present invention is an L-histidine-producing bacteriumof the Enterobacteriaceae family having increased activity of mutantPRPP synthetase of the present invention. More specifically, thebacterium of the present invention is an L-histidine-producing bacteriumof Enterobacteriaceae family, wherein L-histidine production by thebacterium is enhanced by enhancing an activity of the protein of thepresent invention in the bacterium. More preferably, the bacterium ofthe present invention is an L-histidine-producing bacterium belonging tothe genus Escherichia, wherein L-histidine production by the bacteriumis enhanced by enhancing an activity of the protein of the presentinvention, namely mutant PRPP synthetase, in the bacterium. Morepreferably, the bacterium of present invention contains the DNA encodingthe mutant prsA gene, which is overexpressed by the chromosome or by aplasmid in the bacterium. As a result, the bacterium of the presentinvention has enhanced ability to produce L-histidine.

“Bacterium, which has an ability to produce L-histidine” means abacterium which has an ability to cause accumulation of L-histidine in amedium, when the bacterium of the present invention is cultured in themedium. The L-histidine-producing ability may be imparted or enhanced bybreeding. The term “bacterium, which has an ability to produceL-histidine” used herein also means a bacterium, which is able toproduce and cause accumulation of L-histidine in a culture medium in anamount larger than a wild-type or parental strain, and preferably meansthat the bacterium is able to produce and cause accumulation ofL-histidine in a medium in an amount of not less than 0.5 g/L, morepreferably not less than 1.0 g/L.

Enterobacteriaceae family includes bacteria belonging to the generaEscherichia, Erwinia, Providencia and Serratia. The genus Escherichia ispreferred.

The term “a bacterium belonging to the genus Escherichia” means thebacterium which is classified as the genus Escherichia according to theclassification known to a person skilled in the art of microbiology.Examples of microorganisms belonging to the genus Escherichia used inthe present invention include, but are not limited to Escherichia coli(E. coli).

The phrase “activity of the mutant PRPP synthetase is enhanced” meansthat the activity per cell is higher as compared to a non-modifiedstrain, for example, a wild-type strain. For example, this meaningincludes increasing the number of mutant PRPP synthetase molecules percell, increasing the specific activity per mutant PRPP synthetasemolecule, and so forth. Furthermore, Escherichia coli K-12 is an exampleof a wild-type strain that may serve as control. As a result ofenhancement of intracellular activity of the mutant PRPP synthetase, anincrease in the amount of L-histidine accumulation in a medium isobserved.

Enhancement of the mutant PRPP synthetase activity in a bacterial cellcan be attained by enhancement of expression of a gene coding for themutant PRPP synthetase. The mutant PRPP synthetase gene of the presentinvention may encompass any of the genes encoding mutant PRPP synthetasederived from bacteria of Enterobacteriaceae family as well as the genesderived from other bacteria such as coryneform bacteria. Among these,genes derived from bacteria belonging to the genus Escherichia arepreferred.

Transformation of a bacterium with a DNA encoding a protein meansintroduction of the DNA into a bacterium cell using conventionalmethods. As a result, expression of the gene encoding the protein ofpresent invention is increased and the activity of the protein isenhanced in the bacterial cell.

Methods of enhancement of gene expression include increasing the genecopy number. Introduction of a gene into a vector that is able tofunction in a bacterium belonging to the genus Escherichia will increasethe copy number of the gene. For such purposes, multi-copy vectors arepreferably used. The multi-copy vector is exemplified by pBR322, pUC19,pBluescript KS⁺, pACYC177, pACYC184, pAYC32, pMW119, pET22b or the like.Other methods of gene expression enhancement can be achieved byintroduction of multiple copies of the gene into the bacterialchromosome by, for example, methods of homologous recombination, or thelike.

Other methods of gene expression enhancement can be achieved by placingthe DNA of the present invention under the control of a more potentpromoter instead of the native promoter. The strength of a promoter isdefined by the frequency of RNA synthesis initiation. Methods forevaluating the strength of a promoter and examples of potent promotersare described by Deuschle, U., Kammerer, W., Gentz, R., Bujard, H.(Promoters in Escherichia coli: a hierarchy of in vivo strengthindicates alternate structures. EMBO J. 1986, 5, 2987-2994). Forexample, the P_(R) promoter is known as a potent constitutive promoter.Other known potent promoters are P_(L) promoter, lac promoter, trppromoter, trc promoter, of lambda phage and the like.

The enhancement of translation can be achieved by introducing a moreefficient Shine-Dalgarno sequence (SD sequence) into the DNA of thepresent invention in place of the native SD sequence. The SD sequence istypically a region upstream of the start codon of the mRNA interactingwith the 16S RNA of ribosome (Shine J. and Dalgarno L., Proc. Natl.Acad. Sci. U S A, 1974, 71, 4, 1342-6).

Use of a more potent promoter can be combined with multiplication ofgene copies.

Preparation of chromosomal DNA, hybridization, PCR, plasmid DNApreparation, DNA digestion and ligation, transformation, selection of anoligonucleotide as a primer, and the like, are all methods well known toone skilled in the art. These methods are described in Sambrook, J., andRussell D., “Molecular Cloning A Laboratory Manual, Third Edition”, ColdSpring Harbor Laboratory Press (2001), and the like.

The bacterium of the present invention can be obtained by introductionof the aforementioned DNAs into a bacterium inherently having theability to produce L-histidine. Alternatively, the bacterium of presentinvention can be obtained by imparting an ability to produce L-histidineto a bacterium already containing the DNAs.

The parent strain to be enhanced in activity of the protein of thepresent invention includes but is not limited to a bacteria belonging tothe genus Escherichia having L-histidine producing ability, theL-histidine producing bacterium strains belonging to the genusEscherichia, such as E. coli strain 24 (VKPM B-5945, Russian patent2003677); E. coli strain 80 (VKPM B-7270, Russian patent 2119536); E.coli strains NRRL B-12116-B12121 (U.S. Pat. No. 4,388,405); E. colistrains H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat. No.6,344,347); E. coli strain H-9341 (FERM BP-6674) (European patentapplication 1085087A2); E. coli strain A180/pFM201 (U.S. Pat. No.6,258,554), and the like.

It is preferable that the L-histidine-producing bacterium be furthermodified to have enhanced expression of L-histidine biosynthesis. Geneseffective for L-histidine biosynthesis include hisG gene and genes ofhisBHAFI operon, preferably hisG gene encoding ATP phosphoribosyltransferase wherein feedback inhibition by L-histidine is desensitized(Russian patents 2003677 and 2119536).

(3) Method of the Present Invention.

The method of present invention includes a method for producingL-histidine, including the steps of cultivating the bacterium of thepresent invention in a culture medium, allowing the L-histidine toaccumulate in the culture medium, and collecting the L-histidine fromthe culture medium.

In the present invention, the cultivation, collection, and purificationof L-histidine from the medium and the like may be performed byconventional fermentation methods for producing amino acids using amicroorganism. The medium used for culture may be either synthetic ornatural, so long as the medium includes a carbon source, a nitrogensource, minerals and, if necessary, appropriate amounts of nutrientswhich the microorganism requires for growth. The carbon source mayinclude various carbohydrates such as glucose and sucrose, and variousorganic acids. Depending on the mode of assimilation used by the chosenmicroorganism, alcohol including ethanol and glycerol may be used. Thenitrogen source may include various ammonium salts, such as ammonia andammonium sulfate, other nitrogen compounds, such as amines, a naturalnitrogen source, such as peptone, soybean-hydrolysate, and digestedfermentative microorganisms. Minerals may include potassiummonophosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, calcium chloride, and the like. Some additionalnutrients may be added to the medium, if necessary. For instance, if themicroorganism requires proline for growth (proline auxotrophy), asufficient amount of proline may be added to the cultivation medium.

The cultivation is performed preferably under aerobic conditions such asa shaking culture, and stirring culture with aeration, at a temperatureof 20 to 42° C., preferably 37 to 40° C. The pH of the culture isusually between 5 and 9, preferably between 6.5 and 7.2. The pH of theculture can be adjusted with ammonia, calcium carbonate, various acids,various bases, and buffers. Usually, a 1- to 5-day cultivation leads toaccumulation of the target L-amino acid in the liquid medium.

After cultivation, solids such as cells can be removed from the liquidmedium by centrifugation or membrane filtration, and then the targetL-amino acid can be collected and purified by ion-exchange,concentration and crystallization methods.

EXAMPLES

The present invention will be more concretely explained below withreference to the following non-limiting examples. In the examples, anamino acid is of L-configuration unless otherwise noted.

Example 1 Cloning of the Wild-Type prsA Gene from E. coli andConstruction of the Mutant prsDA and prsDS Genes

The entire nucleotide sequence of E. coli strain K-12 has been reported(Science, 277, 1453-1474, 1997). Based on the reported nucleotidesequence, the primers depicted in SEQ ID No. 3 (primer 1) and SEQ IDNo.4 (primer 2) were synthesized and used for amplification of prsAgene. The primer 1 contains a BglII recognition site introduced at the5′ thereof. The primer 2 contains a XbaI recognition site introduced atthe 5′-end thereof.

Chromosomal DNA of E. coli K12 was used as template for PCR, and wasprepared by an ordinary method. PCR was carried out on the AppliedBiosystems GeneAmp PCR System 2400 under the following conditions:initial DNA denaturation at 95° C. for 3 min; followed by 30 cycles ofdenaturation at 95° C. for 30 sec, annealing at 60° C. for 60 sec andelongation at 72° C. for 120 sec; and the final polymerization for 7 minat 72° C. using Taq polymerase (Fermentas, Lithuania). The resulting PCRfragment containing prsA gene without a promoter was treated with BglIIand XbaI and inserted under P_(R) promoter into the integrative vectorpMW 119-P_(R) previously treated with the same enzymes. VectorpMW119-P_(R) was constructed from a commercially available vector pMW119by insertion of P_(R) promoter from phageλ and attR and attL sitesnecessary for further Mu-integration. Thus, plasmid pMW-P_(R)-prsA wasobtained.

Mutant prsDA gene (substitution of aspartic acid 128 with alanine in thePRPP synthetase coded by mutant prsDA gene) was obtained by PCR asdescribed above using primers 1 (SEQ ID No. 3) and 2 (SEQ ID No. 4), andusing plasmid pUCprsDA as a template. Plasmid pUCprsDA is described indetail in the European patent application EP1004663A1. The resulting PCRproduct was treated with BglII and XbaI and inserted under the controlof the P_(R) promoter into the integrative vector pMW119-P_(R)previously treated with the same enzymes. Thus, plasmid pMW-P_(R)-prsDAwas obtained.

Mutant prsDS gene (substitution of aspartic acid 115 with serine in thePRPP synthetase coded by mutant prsDS gene) was constructed by twosuccessive PCR runs. First, two fragments of the gene were synthesizedusing primers 1 (SEQ ID No. 3) and 3 (SEQ ID No. 5) for the firstfragment, and primers 2 (SEQ ID No. 4) and 4 (SEQ ID No. 6) for thesecond one. Chromosomal DNA of E. coli K12 was used as a template. Thenthe resulting PCR products were separated by electrophoresis and elutedfrom gel. In the second PCR run, these two DNA fragments were annealedand the mutant prsDS gene was completed. The resulting PCR fragmentcontaining prsDS gene without a promoter was treated with BglII and XbaIand inserted under the control of the P_(R) promoter into theintegrative vector pMW119-P_(R) previously treated with the sameenzymes. Thus, plasmid pMW-P_(R)-prsDS was obtained.

Example 2 Effect of Enhanced Expression of the purH Gene on HistidineProduction

Three L-histidine-producing plasmid-less strains containing additionalcopies of the prsA, prsDA or prsDS genes integrated into the bacterialchromosome were constructed. The L-histidine producing E. coli strain 80was used as a parental strain for integration of the prsA, prsDA andprsDS genes into the bacterial chromosome. The strain 80 is described inRussian patent 2119536 and deposited in the Russian National Collectionof Industrial Microorganisms (Russia, 113545 Moscow, 1^(st) Dorozhnyproezd, 1) under accession number VRPM B-7270.

Integration of the genes into the chromosome of strain 80 was performedin two steps. For the first step, the histidine-producing strain 80 wastransformed with a helper plasmid containing replicon rep(p15A),transposase gene (genes cts62, ner, A, B from phage Mu-cts) andcontaining Tet^(R) marker. For the second step, the resulting strain wastransformed with plasmid pMW-P_(R)-prsA, pMW-P_(R)-prsDA orpMW-P_(R)-prsDS. For integration of the gene into the chromosome theheat-shocked cells were transferred to 1 ml of L-broth, incubated at 44°C. for 20 minutes, then at 37° C. for 40 minutes, and then were spreadonto L-agar containing 10 μg/ml of tetracycline and 100 μg/ml ofampicillin. Colonies grown within 48 hours at 30° C. were inoculated in1 ml of L broth and incubated for 72 hours at 42° C. in tubes. About 10colonies from every tube were checked for ampicillin and tetracyclineresistance. Colonies sensitive to both antibiotics were tested forpresence of additional copies of the prs gene in the chromosome by PCRusing primers 1 (SEQ ID No 3) and primer 5 (SEQ ID No 7). Primer 5contains a sequence complementary to the attR site of phage Mu. For thatpurpose, a freshly isolated colony was suspended in 50 μl of water andthen 1 μl was subject to PCR. PCR conditions were the following: initialDNA denaturation at 95° C. for 5 minutes; then 30 cycles of denaturationat 95° C. for 30 sec, annealing at 57° C. for 60 sec and elongation at72° C. for 120 sec; the final polymerization at 72° C. for 7 min. A fewof the antibiotic-sensitive colonies tested contained the necessary 1515bp DNA fragment. Thus, strains 80::P_(R)-prsA, 80::P_(R)-prsDA, and80::P_(R)-prsDS were obtained.

For mini-jar batch-fermentation one loop of each strain grown on L-agarwas transferred to L-broth and cultivated at 30° C. with rotation (140rpm) to reach an optical density of culture OD₅₄₀≈2.0. Then 25 ml ofseed culture was added to 250 ml of medium for fermentation andcultivated at 29° C. for with rotation (1500 rpm). Duration of thebatch-fermentation was approximately 35-40 hours. After the cultivation,the amount of histidine which accumulated in the medium was determinedby paper chromatography. The paper was developed with a mobile phase:n-butanol : acetic acid: water=4:1:1 (v/v). A solution of ninhydrin(0.5%) in acetone was used as a visualizing reagent.

The composition of the fermentation medium (pH 6.0) (g/l): Glucose 100.0Mameno 0.2 of TN (NH₄)₂SO₄ 8.0 KH₂PO₄ 1.0 MgSO₄ × 7H₂0 0.4 FeSO₄ × 7H₂00.02 MnSO₄ 0.02 Thiamine 0.001 Betaine 2.0 L-proline 0.8 L-glutamate 3.0L-aspartate 1.0 Adenosine 0.1

Obtained data are presented in the Table 1: TABLE 1 Yield per IntegratedDCW, Histidine, glucose Strain gene g/l g/l (%) 80 — 8.4 16.9 20.4080::P_(R)-prsA prsA 8.6 15.6 19.1 80::P_(R)-prsDA prsDA 7.3 15.8 19.780::P_(R)-prsDS prsDS 8.5 18.4 22.1

As it seen from the Table 1, the use of mutant prsDS gene coding forPRPP synthetase feedback resistant to purine nucleotides improvedhistidine productivity of the E. coli strain 80.

While the invention has been described with reference to preferredembodiments thereof, it will be apparent to one skilled in the art thatvarious changes can be made, and equivalents employed, without departingfrom the scope of the invention. Each of the aforementioned documents,including the foreign priority documents RU2003132412 and RU2004120501,is incorporated by reference herein in its entirety.

1. A mutant bacterial phosphoribosylpyrophosphate synthetase (PRPPsynthetase), wherein the aspartic acid at position 115 in a wild-typephosphoribosylpyrophosphate synthetase from Escherichia coli issubstituted with another L-amino acid residue, and feedback inhibitionby purine nucleotides is desensitized.
 2. The mutant PRPP synthetase ofclaim 1, wherein said aspartic acid residue at position 115 in awild-type PRPP synthetase is substituted with an serine residue.
 3. Themutant PRPP synthetase of claim 2, which includes deletion,substitution, insertion, or addition of one or several amino acids atone or a plurality of positions other than position 115, and whereinfeedback inhibition by purine nucleotides is desensitized.
 4. A DNAencoding said mutant PRPP synthetase of claim
 1. 5. A bacterium of theEnterobacteriaceae family, which contains the DNA of claim 4 and has anability to produce L-histidine.
 6. The bacterium of claim 5, wherein theactivity of said mutant PRPP synthetase is enhanced.
 7. The bacterium ofclaim 6, wherein said bacterium belongs to the genus Escherichia.
 8. Thebacterium of claim 6, wherein the activity of said mutant PRPPsynthetase is enhanced by increasing the expression of said mutant PRPPsynthetase gene.
 9. The bacterium of claim 8, wherein the activity ofsaid mutant PRPP synthetase is enhanced by increasing the copy number ofsaid mutant PRPP synthetase gene, or modifying an expression controlsequence of said gene so that the expression of said gene is enhanced.10. The bacterium of claim 9, wherein the copy number is increased byintegration of additional copies of said mutant PRPP synthetase geneinto the chromosome of the bacterium.
 11. A method for producingL-histidine comprising cultivating the bacterium of claim 5 in a culturemedium, allowing said L-histidine to accumulate in the culture medium,and collecting said L-histidine from the culture medium.
 12. The methodof claim 11, wherein said bacterium has enhanced expression of the genesfor histidine biosynthesis.